Two Pediatric Cases of Primary Ciliary Dyskinesia Caused by Loss-of-Function Variants in Oral-Facial-Digital Syndrome Gene, OFD1

Primary ciliary dyskinesia (PCD) is a hereditary disease caused by genes related to motile cilia. We report two male pediatric cases of PCD caused by hemizygous pathogenic variants in the OFD1 centriole and centriolar satellite protein (OFD1) gene. The variants were NM_003611.3: c.[2789_2793delTAAAA] (p.[Ile930LysfsTer8]) in Case 1 and c.[2632_2635delGAAG] (p.[Glu878LysfsTer9]) in Case 2. Both cases had characteristic recurrent respiratory infections. Neither case had symptoms of oral-facial-digital syndrome type I. We identified a variant (c.2632_2635delGAAG) that has not been previously reported in any case of OFD1-PCD.


Introduction
Primary ciliary dyskinesia (PCD) is a hereditary disorder caused by pathogenic variants of genes related to motile cilia.PCD is characterized by persistent productive cough and is associated with various symptoms including recurrent infections of the upper and lower respiratory tract due to inefcient respiratory mucociliary clearance caused by ciliary beating disorders.Other clinical manifestations include otitis media with efusion, infertility, and left-right laterality defects [1].More than 50 PCD causative genes have been reported and most cases are inherited in an autosomal recessive manner, while X-linked inheritance is rare.OFD1 centriole and centriolar satellite protein (OFD1), a gene on the X chromosome, is associated with oral-facial-digital syndrome type I (OFDSI), which is an X-linked dysmorphology syndrome related to both primary and motile cilia, and is one of the causative genes for X-linked PCD [2].
OFD1 has long been known as the causative gene of primary ciliopathy in OFDSI with a prevalence of 1/50,000 live births [3], and OFDSI is characterized by dysmorphology of the face, oral cavity, and digits with a high degree of phenotypic variability [4].Involvement of the central nervous system is frequent, including central nervous system malformations, mental retardation, and selective cognitive impairment [4].Renal cysts are also common in OFDSI patients, with an overall prevalence of 43%.Renal impairment can be present at birth; however, its prevalence increases with age and most cases develop cystic kidney disease in adulthood [4,5].
In 2006, Budny et al. [6] reported severely uncoordinated and disorganized cilia movement that was observed by highspeed video microscopy of patients' nasal epithelium, which frstly supported the role of OFD1 in respiratory motile ciliary function [6].Patients with OFD1 variants were reported to have features of PCD.Hannah et al. described the frst three PCD cases with pathogenic variants in OFD1 [7].
Here, we report two male Japanese pediatric OFD1-PCD cases.

Case Report
2.1.Case 1.We received a consultation regarding a boy aged 1 year and 7 months with recurrent pneumonia, and blood samples from the case as well as his parents were sent to our department for genetic testing.He was born at 39 weeks.He had nasal obstruction and rhinorrhea immediately after birth.He was not admitted to a neonatal intensive care unit.He had productive cough during the neonatal period and rhinitis that persisted for more than 1 year.He was hospitalized four times due to pneumonia at 2 months, 4 months, 13 months, and 16 months of age, respectively.He did not have situs inversus or a congenital heart defect.He did not have facial dysmorphology or polysyndactyly.His Primary CiliAry DyskinesiA Rule (PICADAR) score [8] was 5 (full-term birth, neonatal respiratory symptoms, and rhinitis), indicating an 11% probability of PCD.
Chest X-ray at 2 months old showed situs solitus and consolidation in the right upper lobe (Figure 1(a)).Chest computed tomography at 3 months of age showed consolidation with an air bronchogram in the right upper lobe (Figure 1(b)).
Transmission electron microscopy of respiratory cilia from the nasal mucosa showed normal ciliary axoneme ultrastructure (Figure 1(c)).His parents and 3-year-old sister did not have PCD or OFDSI.However, three sons of his maternal aunt all had similar clinical symptoms as the proband (they have not undergone genetic testing) (Figure 2

Case 2.
A 5-year-old boy was referred to our department because of persistent wet cough from 1 month of age.He was born at 38 weeks.He was not admitted to a neonatal intensive care unit.He had cough, sputum, and wheezing since 1 month of age.Poor weight gain was noted at 4 months of age.He was hospitalized due to asthma and pneumonia at the age of 3 months and 30 days, and situs inversus was noted at that time.He was hospitalized again at the age of 5 months due to bronchitis.He had secretory otitis media and chronic sinusitis.He did not have facial dysmorphology or polysyndactyly.His PICADAR score was 6 (situs inversus, rhinitis, and otitis media), indicating a 24% probability of PCD.Nasal nitride oxide production was 31.5 nL/min.
Te bilateral tympanic membranes were thickened and the cone of light was absent (Figures 3(a) and 3(b)).Te middle nasal meatus was occluded bilaterally on nasal endoscopy (Figures 3(c) and 3(d)).Chest X-ray at 4 months of age showed situs inversus and increased bronchovascular shadows (Figure 3(e)).Chest computed tomography at 1 year of age showed consolidation with some ground glass opacity in the right lower lobe (Figures 3(f ), 3(g), and 3(h)).
Transmission electron microscopy of respiratory cilia from the nasal mucosa showed the normal ciliary axoneme ultrastructure (Figure 3(i)).His parents and elder brother did not present with any symptoms of PCD or OFDSI (Figure 4

Discussion
We report two male Japanese pediatric PCD cases without associated OFDSI symptoms caused by OFD1.Te variant of Case 1 was the same as that of an OFD1-PCD case reported by Hannah et al. in 2019 [7].Te variant in Case 2 has not been previously reported in any other OFD1-PCD case.Te allele frequency of both variants is unknown.
Te frameshift variant identifed in Case 1 was localized in exon 21 of OFD1 while that of Case 2 was in exon 20.Consistent with these observations, Bukowy-Bieryllo et al. [9] reported four OFD1-PCD patients with truncating variants localized in exons 20 and 21 who all lacked OFDSI symptoms.Te diferent locations of OFD1 variants cause three diferent syndromes: OFDSI (exons 2-17), Simpson-Golabi-Behmel syndrome type 2 (exon 16), and Joubert syndrome 10 (exons 17-22).Te severity of the clinical phenotype generally decreases as variants are localized closer to the C-terminus [9].Te C-terminal part of OFD1, which includes exons 16-23 (amino acids 719-991), contains fve intrinsically disordered regions that are involved in proteinprotein interactions.Te C-terminal part of OFD1 interacts with protein partners and is important for the biogenesis of motile cilia.Changes in the intrinsically disordered regions can afect the binding of OFD1 with its protein partners and consequently lead to motile cilia dysfunction.Bukowy-Bieryllo et al. [9] confrmed that variants in exon 21 do not cause nonsense-mediated decay but may only lead to truncation of the C-terminal part.Tis may explain why the 2 Case Reports in Genetics   Proband (III-2)

A A T T T T A A A A
Father (II-2)

T T T T T A A A A A A A A A
Mother (II-3)   Case Reports in Genetics current two cases did not have OFDSI symptoms but presented with PCD symptoms.Hannah et al. [7] reported three OFD1-PCD cases, and one of the patients carried the same frameshift variant as the present Case 1. Case 1 had inherited this variant maternally while that in the case reported by et al. was a de novo variant [7].Heterozygous mutations of OFD1 are often de novo and approximately 75% of afected females have a de novo variant [5,10].Our Case 2 also had a de novo variant.

Father (II-1) A A A A A A A A A A A A A A
OFD1 protein localizes to the centrosome and basal body of primary cilia [11].An absence of cilia at the embryonic node and laterality defects are observed in male Ofd1 knockout mice [12].Ofd1-defective zebrafsh show a bent body, laterality defects, and edema [13].Tese fndings indicate that Ofd1 is required for primary cilia ciliogenesis and left-right specifcation.Consistent with the literature, our Case 2 also showed situs inversus.
At the centrosomal level, OFD1 is a negative regulator of centriole elongation [14] and interacts with other OFD proteins to initiate ciliogenesis and control centriole length.[15] Knockdown of OFD1 induces hyperelongated centrioles and afects membrane anchoring, leading to the absence of cilia or abnormal cilium length.[16] Bukowy-Bieryllo et al. [9] found that OFD1-PCD patients have longer respiratory cilia than other PCD patients by high-speed video analysis and immunostaining.However, OFD1-PCD patients have normal ciliary axoneme architecture according to axonemal marker staining.Consistent with the study of Bukowy-Bieryllo et al. [9] the two cases reported here showed normal respiratory ciliary axoneme ultrastructure (Figures 1(c) and 3(i)).

Conclusion
We report two male Japanese pediatric PCD cases without associated OFDSI symptoms caused by frameshift variants in exons 20 and 21 of OFD1.In addition, we identifed a variant (c.2632_2635delGAAG) that has not been reported in any OFD1-PCD case.

Figure 1 :
Figure 1: Imaging fndings in Case 1.(a) Chest X-ray at 2 months of age showing situs solitus and consolidation (black arrow) with a loss of lung volume in the right upper lobe.(b) Chest computed tomography showing consolidation (black arrow) with an air bronchogram in the right upper lobe.(c) Transmission electron microscopy fndings of a biopsy specimen from the nasal mucosa of case 1 showing normal ciliary axoneme ultrastructure.Te cilium consists of one pair of central microtubules (white arrow) surrounded by nine pairs of wellarranged peripheral microtubules.Te outer dynein arms (arrowheads) are presented.

Figure 2 :Figure 3
Figure 2: Family pedigree of case 1 with the results of Sanger sequencing.(a) Family pedigree of case 1. III-2 is the proband (Case 1) and his mother (II-3) is a carrier.III-3, -4, and -5 had symptoms similar to those of III-2, but genetic analysis not been performed on these individuals.and II-5 are presumed to be carriers.(b) Te proband (III-2) has a hemizygous variant in OFD1.(c) His father (II-2) does not carry the same variant.(d) Te proband's mother (II-3) is a carrier of the same heterozygous deletion.

Figure 3 :
Figure 3: Imaging fndings in Case 2. Ear endoscopy showing thickened bilateral tympanic membranes without a cone of light in the (a) right and (b) left ears.Te middle nasal meatus is occluded with mucus secretion (white arrows) in the (c) right and (d) left nasal cavities.(e) X-ray at 4 months of age showing situs inversus totalis and increased bronchovascular shadows (black arrow).Chest computed tomography of the (f ) upper, (g) middle, and (h) lower lobes showing consolidation with ground glass opacity (black arrow) in the right lower lobe.(i) Transmission electron microscopy fndings of Case 2. Te cilium consists of one pair of central microtubules (white arrow) surrounded by nine pairs of well-arranged peripheral microtubules.Te outer dynein arms (arrowheads) are presented.

Figure 4 :
Figure 4: Family pedigree of Case 2 with the results of Sanger sequencing.(a) Family pedigree of Case 2. III-2 is the proband.Te parents are not carriers.(b) Te proband (III-2) has a de novo deletion variant in OFD1.(c) Neither his father (II-1) nor (d) mother (II-2) carries the same variant.